The present invention relates to GPI-anchored p97, a secreted form of p97 and derivatives thereof; methods of using p97 in modulating iron transport, in the delivery of drugs, and in the treatment of conditions involving disturbances in iron metabolism; and methods of treating and diagnosing Alzheimer""s Disease.
Iron is a fundamental component required by all cells for growth and normal physiological processes (Crichton, R. R. and Charloteaux-Wauters, M. Eur. J. Biochem. 164:485-506 and Ponka, P. et al, Iron Transport and Storage, CRC Press, Boca Raton, Ann Arbor and Boston, 1990). Rapidly proliferating cells have a higher iron requirement than quiescent cells. In humans this iron requirement is thought to be provided by the binding of iron to the major serum iron-transporting protein, transferrin. Transferrin bound to iron can bind as a complex to the transferrin receptor expressed on the plasma membrane (Ponka, P. et al, Iron Transport and Storage, CRC Press, Boca Raton, Ann Arbor and Boston, 1990). After binding, the iron/transferrin/transferrin receptor complex remains membrane bound and is concentrated and then endocytosed via endocytotic vesicles. The endosomes become acidified and the iron is released from the complex within the cell and the apotransferrin remains bound to the receptor and is recycled to the surface where it is released to participate in the uptake of further iron into the cell (Kuhn L. C. et al., in Iron Transport and Storage, CRC Press, Boca Raton, Ann Arbor and Boston, 1990, p. 149).
Disruption of blood circulation deprives cells of oxygen and iron and may result in cell death. Deposition of iron from cell death, for example in ischemic injury may result in the generation of highly reactive and toxic superoxide or hydroxyl free radicals which can result in further tissue damage. Accordingly, the abundance of iron and its availability can greatly alter survival of damaged tissues. Rapidly proliferating cells, such as malignant cells, have an increased requirement for iron and must possess efficient mechanisms to obtain iron. Limiting the ability of malignant cells to acquire iron may provide a method of killing tumor cells or of modulating their uncontrolled cell growth.
Fe is rarely found in the blood plasma in the free state since it is highly toxic (Lauffer, R. B. (1992). Iron and Human Disease (Boca Ranton, Fla.: CRC Press)) and Tf serves mainly to mop up free Fe and to shuttle Fe, in a soluble non-toxic form, among the organs of the body. The established mechanism by which cells acquire Fe from Tf involves Tf binding to the transferrin receptor (TR) and Fe being internalized by the mechanism of receptor mediated endocytosis(RME) (Aisen, P. (1989). Iron carriers and iron proteins. In Iron carriers and iron proteins. T. M. Loehr, ed. (New York: VCH), pp. 353-372.; Thorstensen, K. and Romslo, I. Biochem. J., 271, 1-10, 1990). Since normal levels of serum Tf are high and about 99% of Fe in the plasma is bound to Tf (May, P. M. et al, (1980). Biological significance of low molecular weight iron(III) complexes. In Metal ions in biological systems. H. Sigel, ed. (New York: Marcel Dekker Inc.), pp. 29-76), Fe uptake is believed to be regulated by the level of TR expression (Thorstensen, K. and Romslo, I. Biochem. J., 271, 1-10, 1990; Young, S. P. and Aisen, P. Hepatology, 1, 114-119, 1981; Brissot, P. et al., J.Clin.Invest., 76, 1463-1470, 1985). Any free Fe generally circulates as low molecular weight complexes such as citrate (Grootveld, M. et al., J. Biol. Chem., 264, 4417-4422, 1989) and certain amino acids or in association with other serum proteins such as albumin (May, P. M. et al, (1980). Biological significance of low molecular weight iron(III) complexes. In Metal ions in biological systems. H. Sigel, ed. (New York: Marcel Dekker Inc.), pp. 29-76). High levels of free Fe are usually only found in the plasma from dying cells or during iron overload disorders such as haemochromatosis (Smith, L., West. J. Med., 153, 296-308, 1990), thalassaemia (Modell, B. and Berdoukas, V. (1984). The clinical approach to thalessemia (New York: Grune and Stratton).) and atransferrinanemia (Kaplan, J. et al, J. Biol. Chem., 266, 2997-3004, 1991).
Based on studies where cells were grown in serum free, hence Tf-free, media and in cases of iron overload disorders it has become evident that some cells are able to obtain Fe independent of Tf and the RME pathway.
Although cellular iron uptake has been shown to be mediated mainly by the transferrin receptor (Doering, T. L. et al, J. Biol. Chem. 265:611-614, (1990), a non-transferrin-mediated pathway has been implicated for iron incorporation into cells, including leukemic cells (Basset, P. et al, Cancer Res. 46:1644-1647, 1986), HeLa cells (Sturrock, A. et al, J. Biol. Chem. 265:3139-3145, 1990), hepatocytes (Thorstensen, K., J. Biol. Chem. 263:16837-16841, 1988) and melanoma cells (Richardson, D. R. and Baker, E., Biochem. Biophys. Acta. 1053:1-12, 1990; Richardson, D. R. and Baker, E., Biochem. Biophys. Acta. 1091:294-302, 1991a and; Richardson, D. R. and Baker, E., Biochem. Biophys. Acta. 1093:20-28, 1991a).
p97, also known as melanotransferrin, a human melanoma-associated antigen, was one of the first cell surface markers associated with human skin cancer (Hellstrom, K. E. and Hellstrom, I. (1982) in Melanoma Antigens and Antibodies, Ed. Reisfield, R. and Ferrone, S., Plenum Press, New York, pp187-341). p97 is a monomeric membrane-associated protein with a molecular mass of 97,000 daltons (Brown, J. P. et al. J. Immunol. 127:539, 1981) and has been suggested as a melanoma specific marker (Estin, C. D. et al., Proc. Nat. Acad. Sci. U.S.A. 85:1052-1056, 1988). As well as being associated with the cell surface of melanomas and some other tumors and cell lines (Brown, J. P. et al., Proc. Nat. Acad. Sci. U.S.A. 78:539, 1981), p97 has also been found in certain fetal tissue (Woodbury, R. G. et al., Int. J. Cancer 27:145, 1981) and, more recently on endothelial cells of the human liver (Sciot, R., et al., Liver 9:110, 1989).
The primary structure of p97, deduced from its mRNA sequence indicates that it belongs to a group of closely related iron binding proteins found in vertebrates (Rose, T. M. et al., Proc. Nat. Acad. Sci. U.S.A. 83:1261, 1986). This family includes serum transferrin, lactoferrin and avian egg white ovotransferrin. Human p97 and lactoferrin share 40% sequence homology (Baker, E. N. et al., Trends Biochem. Sci. 12:350, 1987), however, in contrast to the other molecules of the transferrin family, p97 is the only one which is directly associated with the cell membrane. The deduced sequence of p97 has, in addition to a transferrin-like domain, a hydrophobic segment at its C terminal which was thought to allow the molecule to be inserted into the plasma membrane (Rose, T. M. et al., Proc. Nat. Acad. Sci. USA 77:6114, 1980).
Detergent-solubilized p97 has been reported to bind iron (Doering, T. L. et al., J. Biol. Chem. 265:611-614, 1990). However, the role of p97 in iron transport is far from clear. Iron binding to p97 at the plasma membrane has not been demonstrated and, despite numerous studies, no evidence of a role for p97 in iron mediated transport has been obtained to date. Recent studies have concluded that p97 does not play a role in iron transport (Richardson, D. R. and Baker, E. Biochem. Biophys. Acta. 1103:275-280, 1992; Richardson, D. R. and Baker, E. Biochem. Biophys. Acta. 1093:20-28, 1991 and; Richardson, D. R. and Baker, E. Biochem. Biophys. Acta. 1091:294-302, 1991). The physiological role of p97 in normal and malignant cells has not been determined.
Alzheimer""s Disease has become a significant health care problem due to increases in number and longevity of the elderly. In the near future, it is predicted that a significant proportion of the elderly population may be affected. The incidence of Alzheimer""s Disease increases sharply from 1% at age 65, to over 20% at age 80. After age 85, nearly half of the population in the United States meets the diagnostic criteria for Alzheimer""s Disease (Evans et al, J.A.M.A. 262:2551-2556, 1989).
There are two alternative xe2x80x9ccriteriaxe2x80x9d which are utilized to clinically diagnose Alzheimer""s Disease: the DSM-IIIR criteria and the NINCDS-ADRDA criteria (which is an acronym for National Institute of Neurological and Communicative Disorders and Stroke (NINCDS) and the Alzheimer""s Disease and Related Disorders Association (ADRDA); see McKhann et al., Neurology 34:939-944, 1984). Briefly, the criteria for diagnosis of Alzheimer""s Disease under DSM-IIIR include (1) dementia, (2) insidious onset with a generally progressive deteriorating course, and (3) exclusion of all other specific causes of dementia by history, physical examination, and laboratory tests. Within the context of the DSM-IIIR criteria, dementia is understood to involve xe2x80x9ca multifaceted loss of intellectual abilities, such as memory, judgement, abstract thought, and other higher cortical functions, and changes in personality and behaviour.xe2x80x9d (DSM-IIR, 1987).
In contrast, the NINCDS-ADRDA criteria sets forth three categories of Alzheimer""s Disease, including xe2x80x9cprobable,xe2x80x9d xe2x80x9cpossible,xe2x80x9d and xe2x80x9cdefinitexe2x80x9d Alzheimer""s Disease. Clinical diagnosis of xe2x80x9cpossiblexe2x80x9d Alzheimer""s Disease may be made on the basis of a dementia syndrome, in the absence of other neurologic, psychiatric or systemic disorders sufficient to cause dementia. Criteria for the clinical diagnosis of xe2x80x9cprobablexe2x80x9d Alzheimer""s Disease include (a) dementia established by clinical examination and documented by a test such as the Mini-Mental test (Foldstein et al., J. Psych. Res. 12:189-198, 1975); (b) deficits in two or more areas of cognition; (c) progressive worsening of memory and other cognitive functions; (d) no disturbance of consciousness; (e) onset between ages 40 and 90, most often after age 65; and (f) absence of systemic orders or other brain diseases that could account for the dementia. The criteria for definite diagnosis of Alzheimer""s Disease include histopathologic evidence obtained from a biopsy, or after autopsy. Since confirmation of definite Alzheimer""s Disease requires histological examination from a brain biopsy specimen (which is often difficult to obtain), it is rarely used for early diagnosis of Alzheimer""s Disease.
Neuropathologic diagnosis of Alzheimer""s Disease is typically based upon the numbers of plaques and tangles in the neurocortex (frontal, temporal, and parietal lobes), hippocampus and amygdala (Khachaturian, Arch. Neurol. 42:1097-1105; Esiri, xe2x80x9cAnatomical Criteria for the Biopsy diagnosis of Alzheimer""s Disease,xe2x80x9d Alzheimer""s Disease, Current Research in Early Diagnosis, Becker and Giacobini (eds.), pp. 239-252, 1990). A diagnosis of Alzheimer""s Disease based upon neuropathologic criteria alone, however, is often difficult because there are a significant number of plaques and tangles in the neurocortex, hippocampus, and amygdala of normal elderly people. In addition, the density of neocortical plaques and tangles has only a rough correlation with the degree of dementia.
Some researchers have suggested the use of quantitative electroencephalographic analysis (EEG) to diagnose Alzheimer""s Disease. This method employs Fourier analysis of the beta, alpha, theta, and delta bands (Riekkinen et al., xe2x80x9cEEG in the Diagnosis of Early Alzheimer""s Disease,xe2x80x9d Alzheimer""s Disease, Current Research in Early Diagnosis, Becker and Giacobini (eds.), pp. 159-167, 1990) in order to arrive at diagnosis of Alzheimer""s Disease. This method, however, produces results which are difficult to interpret without control data (such as a routine EEG) from the very same patient prior to onset of Alzheimer""s Disease.
Other researchers have attempted to diagnose Alzheimer""s Disease by quantifying the degree of neural atrophy, since such atrophy is generally accepted as a consequence of Alzheimer""s Disease. Examples of these methods include computed tomographic scanning (CT), and magnetic resonance imaging (MRI) (Leedom and Miller, xe2x80x9cCT, MRI, and NMR Spectroscopy in Alzheimer""s Disease,xe2x80x9d Alzheimer""s Disease, Current Research in Early Diagnosis, Becker and Giacobini (eds.), pp. 297-313, 1990). Although these methods show promise, they cannot yet be utilized to reliably differentiate Alzheimer""s patients from normal elderly people (Bird, Prog. Neurobiol. 19:91-115, 1982; Wilson et al., Neurology 32:1054-1057, 1982; Yerby et al., Neurology 35:1316-1320, 1985; Luxenberg et al., J. Neurol. Sci. 13:570-572, 1986; and Friedland et al., Ann. Int. Med. 109:298-311, 1988).
Other researchers have noticed that patients with Alzheimer""s Disease often exhibit decreased cerebral blood flow or metabolism in the posterior temporoparietal cerebral cortex. These researchers have therefore attempted to measure decreased blood flow or metabolism by positron emission tomography (PET) (Parks and Becker, xe2x80x9cPositron Emission Tomography and Neuropsychological Studies in Dementia,xe2x80x9d Alzheimer""s Disease""s, Current Research in Early Diagnosis, Becker and Giacobini (eds.), pp. 315-327, 1990), single photon emission computed tomography (SPECT) (Mena et al., xe2x80x9cSPECT Studies in Alzheimer""s Type Dementia Patients,xe2x80x9d Alzheimer""s Disease, Current Research in Early Diagnosis, Becker and Giacobini (eds.), pp. 339-355, 1990), and xenon inhalation methods (Jagust et al., Neurology 38:909-912; Prohovnik et al., Neurology 38:931-937; and Waldemar et al., Senile Dementias: II International Symposium, pp. 399407, 1988). These methods, however, are apparently insensitive to damage in structures such as the hippocampus and amygdala, which are believed to be the sites of damage in the earliest stages of Alzheimer""s Disease""s. Therefore, patients may exhibit significant memory loss, and yet exhibit no abnormalities in cerebral blood flow or metabolism.
Various researchers have also attempted to immunologically diagnose Alzheimer""s Disease (Wolozin, xe2x80x9cImmunochemical Approaches to the Diagnosis of Alzheimer""s Disease,xe2x80x9d Alzheimer""s Disease, Current Research in Early Diagnosis, Becker and Giacobini (eds.), pp. 217-235, 1990). Wolozin and coworkers (Wolozin et al., Science 232:648-650, 1986) produced a monoclonal antibody xe2x80x9cAlz50,xe2x80x9d that reacts with a 68-kDa protein xe2x80x9cA68,xe2x80x9d which is expressed in the plaques and neuron tangles of patients with Alzheimer""s Disease. Using the antibody Alz50 and Western blot analysis, A68 was detected in the cerebral spinal fluid (CSF) of some Alzheimer""s patients and not in the CSF of normal elderly patients (Wolozin and Davies, Ann. Neurol. 22:521-526, 1987). This method, however, is not presently suitable as a definitive method for diagnosing Alzheimer""s Disease because detectable levels of A68 could not be found in all patients with xe2x80x9cprobablexe2x80x9d Alzheimer""s Disease (as defined above).
Some researchers have attempted to identify genetic markers for Alzheimer""s Disease. While genetic abnormality in a few families has been traced to chromosome 21 (St. George-Hyslop et al., Science 235:885-890, 1987), such markers on chromosome 21 have not been found in other families with early and late onset of Alzheimer""s Disease (Schellenberg et al., Science 241:1507-1510, 1988).
Others have attempted to identify neurochemical markers of Alzheimer""s Disease. Neurochemical markers which have been associated with Alzheimer""s Disease include reduced levels of acetylcholinesterase (Giacobini and Sugaya, xe2x80x9cMarkers of Cholinergic Dysfunction in Alzheimer""s Disease,xe2x80x9d Alzheimer""s Disease, Current Research in Early Diagnosis, Becker and Giacobini (eds.), pp. 137-156, 1990), reduced somatostatin (Tamminga et al., Neurology 37:161-165, 1987), a negative relation between serotonin and 5-hydroxyindoleacetic acid (Volicer et al., Arch Neurol. 42:127-129, 1985), greater probenecid-induced rise in homovanyllic acid (Gibson et al., Arch. Neurol. 42:489-492, 1985) and reduced neuron-specific enolase (Cutler et al., Arch. Neurol. 43:153-154, 1986). None of these markers, however, is believed to be sensitive or specific enough to provide an early diagnosis of Alzheimer""s Disease (see Elby, xe2x80x9cEarly Diagnosis of Alzheimer""s Disease,xe2x80x9d Alzheimer""s Disease: Current Research in Early Diagnosis, Becker and Giacobini (eds.), Taylor and Francis (pub.), N.Y., pp. 19-30,1990).
Alzheimer""s Disease has been difficult to not only diagnose, but to treat. The discovery that levels of acetylcholinestease are markedly reduced in the cortex and hippocampus of patients with Alzheimer""s Disease (Bowen et al., Brain 99:459-496, 1976) has resulted in the development of a number of pharmaceutical compounds which restore or replace cholinergic function. Examples of such compounds include tacrine (THA) (Summers et al., N. Eng. J. Med. 315:1241-1245); oral administration of choline and lecithin (Etienne et al. Neurology 31:1552-1554, 1981); huperzine A and B (Tank et al., xe2x80x9cStudies on the Nootropic Effects of Huperzine A and B: Two Selective AChE Inhibitors,xe2x80x9d Current Research in Alzheimer""s Therapy, Giacobini and Becker (eds.), pp. 289-393, 1988); galanthamine (Domino, xe2x80x9cGalanthamine: Another Look at an Old Cholinesterase Inhibitor,xe2x80x9d Current Research in Alzheimer""s Therapy, Giacobini and Becker (eds.), pp. 295-303, 1988); methanesulfonyl fluoride (Moss et al., xe2x80x9cMethanesulfonyl Fluoride: A CNS Selective Cholinesterase Inhibitor,xe2x80x9d Current Research in Alzheimer""s Therapy, Giacobini and Becker (eds.), pp. 305-314, 1988); physostigmine, an irreversible inhibitor of acetylcholinesterase Johns et al., Banbury Report 15:435-449, 1983); and physostigmine derivatives (Brufani et al., xe2x80x9cFrom Physostigmine to Physostigmine Derivatives as New Inhibitors of Cholinesterase,xe2x80x9d Current Research in Alzheimer""s Therapy, Giacobini and Becker (eds.), pp. 343-352, 1988). In general, however, these compounds have met with only limited success.
Given the increasing number of individuals with Alzheimer""s Disease, it is critical that new methods for monitoring and treating the disease be discovered. The present invention provides methods for monitoring Alzheimer""s Disease, as well as methods and compositions for treating Alzheimer""s Disease. These methods and compositions overcome disadvantages of prior methods and compositions, and further provide other related advantages.
The present inventors have surprisingly found that p97 is a GPI-anchored protein. The GPI-anchored protein may be reacted with an enzyme that cleaves at the GPI-anchor to provide a cleaved GPI-anchored p97 protein. The cleaved p97 can be prepared using a novel semi-continuous process. Other cleaved GPI-anchored proteins can also be prepared using the novel semi-continuous process.
The present inventors have also unexpectedly found a soluble form of p97. This soluble form is hydrophilic and is present exclusively in the aqueous phase after Triton-X-114 phase separation; it does not contain ethanolamine, and it has a slower rate of transport than GPI-anchored p97. The soluble form of p97 may be present in biological fluids such as cerebrospinal fluid (CSF), blood, or urine. The present inventors have also shown that p97 is involved in iron transport. GPI-anchored p97 expressed on the cell surface has been shown to bind iron and bound iron is released after phospholipase treatment. Significantly, the present inventors have demonstrated a transferrin independent p97-mediated iron uptake pathway. The presence of p97 was found to double iron uptake into CHO cells.
p97 and transferrin were also found to be expressed in brain capillary endothelial cells in normal controls and pathological brains. Most of the p97 molecule is intracellular and its expression is coincidental with the transferrin receptor. EM also indicates that p97 crosses the blood brain barrier. p97 has also been shown to bind to a soluble form of transferrin receptor. Results of affinity chromatography experiments suggest that there is a receptor which co-recognizes p97 and the transferrin receptor.
These findings suggest that p97 may be used to modulate iron uptake in cells. Iron uptake in cells could be modulated by varying the concentration of p97, inhibiting p97 binding to iron or to the transferrin receptor, or inhibiting binding to the receptor which co-recognizes p97 and the transferrin receptor. Accordingly, p97, and stimulants, agonists or antagonists of p97 may be useful in the treatment of conditions where there is a disturbance in iron metabolism. For example, such substances may be useful in the treatment of conditions such as haemochromatosis, neurodegenerative diseases, ischemic tissue damage, including ischemic stroke or trauma, heart disease, and tumors, in particular skin cancer.
The finding of a role for p97 in iron uptake and iron transport, and in particular the finding that p97 can cross the blood brain barrier, suggests that p97 can be used to transport substances such as therapeutic agents across the blood brain, blood eye or blood placenta barrier.
The present inventors have significantly shown that Alzheimer""s patients have elevated levels of p97 in their serum and cerebrospinal fluid and that p97 levels increase with duration of the disease. The levels of p97 in patient samples may thus be used to diagnose and to monitor the progression of the disease and the efficacy of therapeutic treatments for Alzheimer""s Disease.
The present inventors have also significantly found that reactive microgial cells associated with senile plaques in Alzheimer""s Disease express p97 and transferrin receptor. Therefore, p97 and transferrin receptor can be used in the diagnosis of Alzheimer""s Disease. The finding that microgial cells which deposit the amyloid protein have a high level of proteins which operate in procurement of iron also suggests methods of treatment of Alzheimer""s disease based on depletion of iron from these cells using substances such as p97, transferrin, and iron chelators, for example, lactoferrin, ferritin, ovotransferrin.
Broadly stated the present invention relates to a GPI-anchored form of p97 and derivatives thereof. The invention also contemplates methods of preparing p97 and derivatives thereof.
Within one embodiment of the present invention methods are provided for preparing a cleaved form of the GPI-anchored p97, comprising incubating a cell which expresses p97 on its surface with an enzyme that cleaves glycosyl-phosphatidylinositol (GPI) anchors to produce the cleaved form of the GPI-anchored p97, and isolating the cleaved form. Within the context of the present invention, phospholipase cleaved p97 or cleaved p97 refers to p97 which has been cleaved from its glycosyl-phosphatidylinositol (GPI) anchor.
Preferably, a semi-continuous process for preparing cleaved GPI-anchored proteins such as cleaved GPI-anchored p97 is utilized. The semi-continuous process comprises (a) providing a cell capable of expressing a GPI-anchored protein on its surface; (b) growing the cell under conditions suitable for the expression of the GPI-anchored protein on the cell surface; (c) incubating the cell with an enzyme which is capable of cleaving the GPI anchor to form a cleaved protein; (d) recovering the cleaved protein; and (e) repeating steps (b) to (d) until a desired amount of cleaved protein is obtained. Preferably, the cell is genetically engineered to express the GPI-anchored protein.
Within another aspect of the present invention, isolated soluble p97 is provided. The soluble form of p97 is hydrophilic; present exclusively in the aqueous phase after Triton-X-114 phase separation; it does not contain ethanolamine, and it has a slower rate of transport than GPI-anchored p97. The soluble p97 can be isolated based on its hydrophilic property.
Within yet another aspect of the present invention an isolated DNA sequence is provided which encodes truncated p97. Within various embodiments of the invention, the sequence which encodes truncated p97 consists essentially of the sequence which encodes the C-terminal domain of p97, or the sequence which encodes the N-terminal domain of p97. Also provided are recombinant expression vectors for expressing such sequences, as well as the host cells which contain these expression vectors.
Within one embodiment of the invention, the p97 is labelled, the label being selected from the group consisting of fluorescent molecules, enzymes, luminescent molecules, radionuclides, substances having therapeutic activity, and toxins.
The invention also contemplates methods of modulating iron metabolism using p97. In particular, the present invention relates to a method for treating conditions involving disturbances in iron metabolism comprising administering an iron modulating amount of p97, or a stimulant, agonist or antagonist of p97. Conditions involving disturbances in iron metabolism which may be treated using the method of the invention include haemochromatosis, neurodegenerative diseases, ischemic tissue damage, including ischemic stroke or trauma, heart disease, and tumors, in particular skin cancer.
A substance which is a stimulant, agonist or antagonist of p97 may be identified by determining the effect of the substance on the binding activity of p97 and iron, or p97 and the transferrin receptor, or the effect of the substance on the expression of p97 in cells capable of expressing p97 including cells genetically engineered to express p97 on there surface.
The invention therefore in one aspect relates to a method of identifying stimulants, agonists or antagonists of p97 comprising reacting a substance suspected of being a stimulant, agonist or antagonist of p97 with p97 and iron under conditions such that p97 is capable of binding to the iron; measuring the amount of p97 bound to iron; and determining the effect of the substance by comparing the amount of p97 bound to iron with an amount determined for a control. The invention also relates to a method of identifying stimulants, agonists or antagonists of p97 comprising reacting a substance suspected of being a stimulant, agonist or antagonist of p97 with p97 and transferrin receptor under conditions such that p97 is capable of binding to the transferrin receptor; measuring the amount of p97 bound to transferrin receptor; and determining the effect of the substance by comparing the amount of p97 bound to transferrin receptor with an amount determined for a control.
The invention also relates to a method of identifying stimulants, agonists or antagonists of p97 comprising reacting a substance suspected of being a stimulant, agonist or antagonist of p97 with a cell which expresses p97, measuring the amount of p97 expressed by the cell, and determining the effect of the substance by comparing the amount of expression of p97 with an amount determined for a control.
The invention further relates to a method for identifying a stimulant, agonist or antagonist of p97-mediated iron uptake comprising: incubating a cell expressing p97 on its surface and a substance suspected of being a stimulant, agonist or antagonist of p97 in the presence of iron and in the absence of transferrin, measuring the amount of iron uptake into the cell and identifying a stimulant, agonist or antagonist of p97-mediated iron uptake by comparing the amount of iron uptake in the cell with the amount of iron uptake in a cell from a control incubation in the absence of the substance.
In an embodiment, the cell is incubated in the presence of labelled iron and the amount of iron uptake in the cell is determined by measuring the amount of labelled iron in the cell. The label may be, for example, radioactive or fluorescent.
The invention also relates to a composition for delivering an agent across the blood brain barrier comprising p97 or a substance which is capable of specifically binding to p97, in association with the agent and a pharmaceutically acceptable carrier or diluent. The p97 or substance, preferably antibody to p97 may be conjugated to the agent or a p97 fusion protein may be used in the composition. The agent may be a substance having therapeutic activity such as a growth factor or lymphokine. The invention also relates to a method of delivering an agent across the blood brain barrier comprising administering the agent in association with p97 or antibody to p97. The composition of the invention may also be used for delivering an agent across the blood eye or blood placenta barrier.
Within one aspect of the present invention, a composition for the preservation of organs intended for transplantation is provided comprising p97 or a derivative thereof in a pharmaceutically acceptable organ preservation solution. The invention also contemplates a method for preserving an organ intended for transplantation using the composition.
The present invention also provides methods for diagnosing and monitoring Alzheimer""s Disease, as well as compositions and methods suitable for treating Alzheimer""s Disease. Within one aspect of the present invention, methods are provided for monitoring Alzheimer""s Disease, comprising detecting the presence of soluble p97 in a patient. Within various embodiments, the p97 may be detected in various bodily fluids, including for example, urine, blood, serum and cerebral spinal fluid. Various methods may be utilized to detect p97, including, for example, radioimmunoassays, competitive assays, and enzyme linked immunosorbant assays (ELISA) such as the sandwich assay. Within other aspects of the present invention, methods are provided for monitoring Alzheimer""s Disease comprising detecting the presence of transferrin receptors, and/or detecting the presence of p97, on microglial cells associated with amyloid plaques in a patient.
The invention also provides a method for diagnosing or monitoring Alzheimer""s Disease in a patient, comprising determining the concentration of p97 in a sample from the patient and comparing the determined concentration to the level of p97 in other samples from the patient, control subjects and/or Alzheimer""s Disease patients. The concentration of p97 may be determined by a radioimmunoassay, immunofluorescent assay, competitive assay, or enzyme linked immunosorbant assay. In an embodiment, the sample is a serum sample or a cerebrospinal fluid sample. The sample may be from a patient being monitored to assess the efficacy of a therapeutic treatment, such as the administration of a pharmaceutical composition, for Alzheimer""s Disease.
The invention also contemplates a bispecific antibody capable of binding to a microglial cell which expresses p97 and/or transferrin receptor and to a label preferably a detectable substance, or a substance having toxic or therapeutic activity. The bispecific antibody may be prepared by forming a hybrid hybridoma from a fusion between a first cell line which produces a first monoclonal antibody which is capable of binding to a microglial cell which expresses p97 and/or transferrin receptor and a second cell line which produces a second monoclonal antibody which is capable of binding to the label.
The invention further contemplates a tetrameric immunological complex of a first monoclonal antibody which is capable of binding to a microglial cell which expresses p97 and/or transferrin receptor and a second monoclonal antibody which is capable of binding to a label preferably a detectable substance or a substance having toxic or therapeutic activity wherein the first and second antibody are from a first animal species, conjugated to form a cyclic tetramer with two monoclonal antibodies of a second animal species directed against the Fc-fragment of the antibodies of the first animal species.
The tetrameric immunological complex may be formed by reacting a first monoclonal antibody which is capable of binding to a microglial cell which expresses p97 and/or transferrin receptor and a second monoclonal antibody which is capable of binding to a label preferably a detectable substance or a substance having toxic or therapeutic activity wherein the first and second antibody are from a first animal species, with an about equimolar amount of antibodies of a second animal species which are directed against the Fc-fragments of the antibodies of the first animal species and isolating the tetrameric complex formed.
The bispecific antibodies and tetrameric antibody complexes of the invention when coupled with a detectable substance may be used to identify microglial cells associated with Alzheimer""s Disease.
The present invention also relates to a method of treating Alzheimer""s Disease in a patient comprising depleting iron in the brain, preferably the microglial cells of the patient. In a preferred method of the invention, the treatment comprises administering p97, transferrin, transferrin receptor, or substances which are capable of reacting with p97 or transferrin receptor, preferably antibodies to p97 and transferrin or iron chelators. Exemplary iron chelators are lactoferrin, ferritin, and ovotransferrin.
Within another aspect of the present invention, a method for treating Alzheimer""s Disease is provided comprising the step of administering to a patient labelled p97 or a substance which is capable of binding to p97 conjugated to a label. In one embodiment a labelled antibody to p97, or a bispecfic antibody complex or tetrameric antibody complex specific for a label and p97, and which are conjugated to the label, may be administered. The label may be a toxin selected from the group consisting of ricin, abrin, diptheria toxin, cholera toxin, gelonin, pokeweed antiviral protein, tritin, Shigella toxin, and Pseudomonas exotoxin A.
Within another aspect of the present invention a method for treating Alzheimer""s Disease is also provided comprising the step of administering to a patient a transferrin receptor blocking agent. Examples of transferrin receptor blocking agents include a transferrin receptor blocking antibody, p97 and transferrin. An antibody to the transferrin receptor conjugated to a label as described herein or a bispecfic antibody complex or a tetrameric antibody complex specific for the transferrin receptor and the label, and which is conjugated to the label, may also be used to treat Alzheimer""s Disease.
Within another aspect of the present invention, methods are provided for treating Alzheimer""s Disease comprising administering an antibody which blocks the binding of p97 to iron. Within one embodiment, the antibody is a human antibody.
The invention also contemplates a method of purifying microglial cells associated with Alzheimer""s Disease beta amyloid plaques comprising reacting a sample suspected of containing microglial cells associated with Alzheimer""s Disease beta amyloid plaques with a substance which is capable of specifically binding p97 or transferrin receptor under conditions such that the microglial cells bind to the substance; and isolating the microglial cells bound to the substance. The isolated cells may be transformed to produce a cell line. The cell line may be used to test for substances which affect the microglial cells associated with Alzheimer""s Disease beta amyloid plaques. Accordingly, substances may be identified which are effective in the treatment of Alzheimer""s Disease.
These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, reference is made herein to various publications, which are hereby incorporated by reference in their entirety.